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BSCI 1510L Literature and Stats Guide: 4 Previous work in the BSCI 111 class

Introduction to Biological Sciences lab, first semester

4.1 In vitro experiment

In an early experiment that preceded the research project, fixed amounts of bacteria were exposed to soaps under standardized laboratory conditions rather than attempting to treat bacteria present on hands followed by sampling of the hands.

4.1.1 Protocol (as the experiment was carried out -we will NOT actually do this!)

1. Label five microcentrifuge tubes as follows: Cd (control diluted), Ru (regular soap undiluted), Rd (regular soap diluted), Au (antibacterial soap undiluted), and Ad (antibacterial soap diluted).  Use a serological pipette to place 10 ml of sterile TE buffer in a sterile 15 ml screwcap tube.  Vortex the E. coli stock culture briefly to resuspend the bacteria before removing any of the suspension!   Use your P20 pipetter to transfer 10 µl of the E. coli suspension into the sterile TE.  Close the screwcap tube and vortex it vigorously to mix the suspension.  Use your P1000 pipetter to measure 1.0 ml of diluted E. coli suspension into the Cd, Rd, and Ad microfuge tubes.  Measure 1.0 ml of undiluted stock E. coli suspension into the Ru and Au microfuge tubes.  Close the caps tightly and spin in a microcentrifuge for 20 seconds to pellet the cells.  Be sure the centrifuge is balanced!  You can achieve this by spinning with another group so that there is an even number of tubes in the centrifuge.  If you must spin by yourself, use a tube containing 1 ml of water to balance the fifth tube. 

Note: When microfuging a precipitate, the pellet may be so small that it is not easily seen.  Careful placement of the tube in the microfuge can help solve this problem.  If you place the tube so that the hinge of the cap is oriented in a certain direction (e.g. toward the center), then you will know that the pellet will be located a short distance up from the bottom of the tube on the wall (see image on left) even if you can't see it.  Think about how the tube is oriented in the microfuge as it spins and this will make sense.  Note: if the solution is dilute, you will probably not be able to see the pellet.  Assume that it is there and continue on with the protocol. 

2. Pour off the supernatant into the waste container on your bench.  To the tube labeled "Cd", add 1.0 ml of TE buffer.  Resuspend the soap stocks by inverting.  To the tubes labeled "Ru" and "Rd", add 1.0 ml of regular liquid soap (previously diluted with TE buffer by the lab staff).  To the tubes labeled "Au" and "Ad", add 1.0 ml of antibacterial liquid soap (also previously diluted with TE buffer).  Randomly select one of the five tubes and resuspend the pellet using a vortexer.  Be sure that the entire pellet is resuspended.  In random order, resuspend the pellets in the other four tubes as well.

 

3. Place all five tubes in the floating rack in the 45°C water bath for 1 minute.  (This is to simulate the use of hot water in hand washing.)  Remove the tubes and spin for 20 seconds in a microcentrifuge.  Pour off the supernatent.  Be sure to remove as much soap as possible.

4. Add 1.0 ml of TE buffer to all five tubes.  Randomly select a tube and resuspend the pellet using a vortexer as before.  Repeat with the other four tubes.  Centrifuge and pour off the supernatant.  Add 1.0 ml of TE buffer to all five tubes and resuspend as you did before.  Centrifuge and pour off the supernatant.  Add 1.0 ml of TE buffer to all five tubes and resuspend as you did before, then proceed to the next step.

5. Using a Sharpie marker, label your five plates on the bottoms (not the lids) with your names and section.  Label the plates as follows: "control diluted", "regular undiluted", "regular diluted", "antibacterial undiluted", and "antibacterial diluted".  Remove 100 µl of suspension from a tube using a P200 pipetter and distribute it over the surface of the agar on its corresponding plate.  Use a sterile disposable spreader to spread the suspension evenly over the surface of the plate.  Spread thoroughly using the technique demonstrated by your TA.  If you do not spread thoroughly, the resulting uneven distribution of bacterial colonies will be difficult to count.  Replace the lid and repeat with the other four plates. 

6. Tape your five plates together in a stack using masking tape.  You do not need an excessive amount of tape!  Use just enough on the sides to hold the plates together.  Do not wrap the tape around and around the plates.  Place your stack upside down in the wire basket for your section.  (Incubating the plates upside down prevents condensation on the lid from dripping down and smearing the colonies on the plate.) 

7.  Your plates will be incubated overnight in a 37°C oven.  On the following day (Monday if you are in Friday's section), return to the lab and examine your plates.  If the surface of the agar is covered with a solid lawn of bacteria (Fig. 5 of previous experiment), record this and do not attempt to count colonies.  If the plate contains distinct colonies (Fig. 6 of previous experiment), use a colony counter to count them by one of the following methods.  (A colony results from the radial growth of a single bacterium.  Thus counting the visible colonies produces a measure of the number of bacteria present in the solution plated.)  If there are a few colonies (about 100 or less) on the entire plate, count every colony.  If there are an intermediate number of colonies (100 to 400), use the grid on the counter to divide the plate into quarters.  Count the number of colonies in ¼ of the plate, then multiply by 4 to estimate the total number of colonies on the plate.  If there are a large number of colonies (greater than about 400, Fig. 6 of previous experiment), count the number of colonies in a certain number of representative 1 cm squares (use the grid on the counter as a guide), then use the area of the plate (pr2) to estimate the total number of colonies on the plate.  (Think about how to do this!)  Rulers are available in the lab drawers.  Record your results in your lab notebook, then discard your plates in an autoclave bag.

8.  For the control, antibacterial, and regular soap treatments, calculate the titer of surviving bacteria that would have been present in 1ml of the original stock culture.  You will probably have only one countable trial for each type of treatment.  Do not attempt to calculate a titer for cultures that were lawns.  If the more dilute trial of a treatment had no surviving colonies, then perform the calculation for the more concentrated treatment.  If the more concentrated treatment also had no colonies, then report the titer as zero.  Each colony that you counted represents one surviving bacterium.  However, you did not plate all of the bacteria that you treated, and in some cases the stock was diluted before the treatment.  To compensate for this, divide your count by the dilution factor for any dilutions and by the volume in ml of treated culture that you plated.  Report your titer on the appropriate data sheet by the colony counters.  Use scientific notation if the values are greater than 1 x 106.

4.1.2 Problems with the in vitro protocol as originally designed

For the first several years that this experiment was performed, the results seemed to be clear-cut: all treatments involving antibacterial soap produced few or no colonies, while the regular soap treatments did not seem to differ from the controls which were exposed to buffer only.  However, careful observation of the plates often showed that bacterial colonies were present along the edge of the plates just beyond the visible line where the treated suspension was spread.  This lead to the hypothesis that the one-minute exposure to the antibacterial soap was having little effect on the bacteria and that the actual effect of the treatment was inhibition of colony growth due to the presence of residual triclosan in the treated suspension.  To test this hypothesis, an additional TE buffer rinse was added in step 4.  After this protocol modification was put into place, there was virtually never any effect of either type of soap (counts were not different from the controls).

4.1.3 Lack of realism of the experiment

The lack of apparent effect of the soaps in vitro led to questions about the relevance of a method that did not include the physical removal of bacteria facilitated by the soap during the rubbing and rinsing of hands.  An informal experiment to test the soaps under actual hand-washing conditions was performed by several TAs as described below.

4.2 Finger stamping as an assay method

The Leyden et al. (1991) paper suggested that pressing skin against an agar plate might be a suitable method for assaying the number of bacteria on hands.

4.2.1 An early attempt to measure bacterial numbers using finger stamping

One subject's hands were stuck into soil and rubbed vigorously together to distribute the dirt evenly on the fingers of both hands.  The other subject's hands were left unwashed after working in the BSCI 111 lab for an afternoon (Fig. 1).

Fig. 1 Fingertips of both types of hands were rubbed on two sides of a nutrient agar plate (left).  The unwashed lab hand produced a few bacterial colonies and the unwashed soiled hand produced a wide variety of bacterial and fungal colonies.

 

One hand of each subject was washed with Dial liquid antibacterial soap and one hand was washed with SoftSoap regular liquid soap.  The two hands were washed for the same amount of time in a way that the participants thought was typical.  They were then immediately rinsed with warm running water. 

 

 A finger from each hand was then streaked across the two sides of the plate.  The plate was incubated overnight at 30°C.

       

Results:

Fig. 2  Hands placed in soil

Fig. 3  Hands from lab work

Comparison of Fig. 1 to Figs. 2 and 3 seems to show a fairly clear decrease in the number of colonies caused by washing.  Comparison of the left and right sides of Fig. 2 and Fig. 3 fails to suggest that use of the antibacterial soap produced any benefit over the regular soap.  These results support the idea that the physical effects of washing are more important than the chemical bactericidal effects of the antimicrobial agents. 

The ad hoc nature of this experiment makes it unsuitable for drawing any firm conclusions.  Some of the flaws of the experiment were: lack of replication, lack of standardized hand-washing protocol, and lack of a standardized method to dirty the hands.  However, it demonstrated the feasibility of using a sampling method that involves direct application of hands to an agar plate.

4.2.2 Subsequent experience with finger stamping as an assay method

4.2.2.1 Dampness of fingers

Additional tests showed that the finger stamping method was extremely sensitive to the level of moisture of the skin.  A finger which produced few colonies would subsequently produce many colonies after being dampened with sterile buffer.  

On the top are dry thumbs and on the bottom are thumbs dampened with water.

4.2.2.2 Artificial contamination

Although ambient bacteria on a subject's fingers usually produced a countable number of colonies, it was very difficult to adjust the titer of a bacterial culture to be used for artificial contamination.  The most common outcome of stamping by artificially contaminated fingers was a lawn.  

The stock suspension here is not the same as the standard titer that we will be giving to you.  The point is that it is very difficult to adjust the titer so that the resulting colonies don't overlap.  (The light spots in the upper right are condensation, not colonies.)

4.2.2.3 Ambient bacteria vs. colonizing flora

Repeated washing episodes did not result in elimination of colonies resulting from ambient bacteria.  This implied that ambient bacteria that were resident in deeper skin layers were simply being exposed following the washing episodes.  Thus changes in colony counts after washing in stamping trials were quite different when ambient bacteria were sampled versus when bacteria were added.

Stamping multiple times with the same thumbs:

On the top are dampened thumbs and on the bottom are the same thumbs dampened again and pressed onto a second plate (no washing treatment).